Familial cylindromatosis (MIM 132700) is a syndrome characterized by multiple benign neoplasms of the skin appendages. The tumors are known as cylindromas because of their characteristic microscopic appearance and are thought to resemble the eccrine (sweat gland) or apocrine (scent glands) cells of the skin. Hairy areas of the body are usually affected, with most tumors appearing on the head and neck. Genetic linkage analysis of numerous families has localized the gene to chromosome 16q12-13. Over 70% of familial cylindromas exhibit loss of heterozygosity (LOH) at this locus and in all cases the allele lost is the wild-type allele. Thus, this locus, CYLD1, behaves as a classic tumor suppressor. CYLD1 encodes a 956 amino acid protein with close homologs in other mammals, insects and C. elegans. Numerous mutations in the C-terminal two-thirds of the protein result in truncations and disease. CYLD contains four recognizable sequence motifs: three CAP-GLY domains thought to be involved in binding to microtubules anchoring chromosomes or endocytic vesicles to the cytoskeleton, a putative metal binding deubiquitinating enzymes (Wilkinson and Hochstrasser, 1998). The mechanism by which these mutations cause disease is unknown, but we hypothesize that this protein is a deubiquitinating enzyme and that enzymatic activity is required for tumor suppression. Specific aim 1. We will test the hypothesis that CYLD is an active deubiquitinating enzyme. We will express and purify the protein from bacteria, yeast, or COS cells and measure the rate of hydrolysis of authentic deubiquitinating enzyme substrates. The kinetic parameters will be determined and specificity for ubiquitin will be evaluated by using a panel of ubiquitin homologs. Specific aim 2 will test the hypothesis that CYLD acts on polyubiquitin chains. The ability of CYLD (purified as above) to hydrolyze polyubiquitin chains will be determined. Chains linked through K48, K63 or K29 will be used as substrates. Binding to non-hydrolyzable chains will also be tested.